Genetic evidence for algal auxin production in Chlamydomonas and its role in algal-bacterial mutualism

Summary Interactions between algae and bacteria are ubiquitous and play fundamental roles in nutrient cycling and biomass production. Recent studies have shown that the plant auxin indole acetic acid (IAA) can mediate chemical crosstalk between algae and bacteria, resembling its role in plant-bacterial associations. Here, we report a mechanism for algal extracellular IAA production from L-tryptophan mediated by the enzyme L-amino acid oxidase (LAO1) in the model Chlamydomonas reinhardtii. High levels of IAA inhibit algal cell multiplication and chlorophyll degradation, and these inhibitory effects can be relieved by the presence of the plant-growth-promoting bacterium (PGPB) Methylobacterium aquaticum, whose growth is mutualistically enhanced by the presence of the alga. These findings reveal a complex interplay of microbial auxin production and degradation by algal-bacterial consortia and draws attention to potential ecophysiological roles of terrestrial microalgae and PGPB in association with land plants.


INTRODUCTION
The auxin indole-3-acetic acid (IAA) is an essential signaling molecule that controls almost every aspect of plant development. 1,2][5][6][7][8] As in land plants, IAA plays a key role in controlling growth, photosynthetic activity, and primary metabolism of microalgae. 5,7,9,10Some bacteria produce IAA extracellularly to stimulate algal photosynthetic activity, primary metabolism, and growth. 5,9,11,124][15][16][17][18][19] Nevertheless, the molecular basis for how IAA is made in algae and its potential role in interkingdom interactions remains essentially unknown. 17,20,21he ''phycosphere,'' the algal analog of the plant rhizosphere, is the region immediately surrounding an algal cell that is enriched in exuded metabolites similar to those released by plant roots. 5,22,23These compounds include IAA and nutrients such as sugars and amino acids that serve ecological roles, are exchanged with bacteria, and act as signaling molecules essential for communication between taxa. 12There is mounting evidence of conspicuous similarities between the bacterial associations formed with algae and those formed with land plants 24 (reviewed by Seymour et al. 25 ).For instance, many bacteria that associate with algae, such as Rhizobium and Sphingomonas, are phylogenetically similar to those that form symbioses with land plants, suggesting broad eco-evolutionary affinities between bacteria and the diverse lineages of the plant kingdom. 24,26,27Some of these beneficial interactions have been exploited for biotechnological applications, [28][29][30] notably toward improving sustainable agriculture. 31,32However, we still understand surprisingly little about both the ecology and the molecular mechanisms underlying most algal-bacterial interactions in nature. 25,33,34n prior work, we found that the model alga Chlamydomonas reinhardtii 35 and plant-growth-promoting bacteria (PGPB) in the genus Methylobacterium 36 can form mutualisms based on a carbon (C) and nitrogen (N) nutrient exchange. 37This beneficial interaction between Chlamydomonas and Methylobacterium can be harnessed to improve wastewater bioremediation, biomass generation, and hydrogen production. 38Briefly, these bacteria can mineralize exogenous amino acids and peptides that are poor N sources for Chlamydomonas to support the growth of the alga, which in turn provisions photosynthetic C sources like glycerol to promote bacterial growth. 370][41] LAO1 is highly expressed in Chlamydomonas during N starvation and oxidizes L-amino acids present in the extracellular medium to produce ammonium, hydrogen peroxide, and corresponding keto acids 41 (Table S1).Although ammonium is used to support algal growth, the resulting keto acids are surprisingly not used as a C source by the alga. 39,40,42We reasoned that although not metabolized by Chlamydomonas, these extracellular keto acids may provide some benefit and/or play some ecological role.For instance, some aminoacid-derived keto acids like a-ketoisocaproic (from L-leucine) or a-ketoisovaleric (from L-valine) show siderophore activity that may improve iron nutrition; 43 others, like pyruvate (from L-alanine) and oxaloacetate (from L-aspartate), can scavenge hydrogen peroxide 44 to potentially reduce oxidative stress; still others may serve as carbon sources for nearby organisms.Some LAO1-derived keto acids like indole pyruvic acid (IPyA, from L-tryptophan [L-Trp]) and phenyl pyruvic acid (PPA, from L-phenylalanine) are well-known precursors for auxin biosynthesis. 45,46onetheless, LAAO-mediated production of auxin has only recently been identified in a single bacterial species. 47We surmise that LAAO enzymes may play significant ecological roles in mediating cross-taxa interactions, particularly between microbes and diverse plant lineages, despite their relevance being largely neglected to date.
In this work, we present the first ever genetic evidence for auxin production in an alga, specifically the model alga Chlamydomonas.IAA is accumulated extracellularly under N limitation in an LAO1-dependent manner, expanding the role of LAAOs in microbial auxin production.We show the impact of extracellular IAA accumulation under N limitation on Chlamydomonas growth and its potential role in establishing beneficial interactions with methylobacteria.Our study reveals a new mode of algal-bacterial interaction mediated by algal-produced IAA that may have fundamental implications about the ecological impact of algae and PGPB in aquatic systems and terrestrial ecosystems potentially modulating plant responses. 48

RESULTS
Chlamydomonas produces IAA from tryptophan using the extracellular L-amino acid oxidase LAO1 L-tryptophan (L-Trp) is the major substrate for IAA production in bacteria, fungi, algae, and land plants (reviewed by Morffy et al. 20 ).Chlamydomonas can transform exogenous L-Trp and other amino acids into ammonium and a corresponding a-keto acid by means of the extracellular deaminase LAO1. 39The LAO1 gene is highly induced by nitrogen (N) limitation. 41,49This enzyme supports growth on L-Trp as the sole N source; lao1 knockout mutants completely lose the ability to grow under these conditions (see Calatrava et al. 41 ; supported by Figure S1A).To test whether Chlamydomonas LAO1 is involved in IAA biosynthesis, log-phase wild-type (WT) and lao1 null mutant cells were incubated for 48 h in growth medium supplemented with 5 mM L-Trp as a sole source of N (Figure 1).The concentrations of L-Trp, the corresponding ketoacid indole-3-pyruvic acid (IPyA), and IAA were quantified in cell-free supernatants by HPLC (Figures 1A-1C, respectively), and the identity of these compounds was confirmed by LC-MS/MS (Figures 1D-1F).WT cultures depleted L-Trp and accumulated the byproducts IPyA and IAA in the medium, along with other IAA biosynthesis intermediates; indole-3-acetamide and indole-3-acetaldehyde were also present in the media but did not accumulate over time.The L-Trp metabolism product kynurenic acid also accumulated in the media (Figures S2-S5).The media of the lao1 mutant cultures and the controls without cells did not accumulate IAA and IPyA, and the L-Trp concentration remained constant (Figures 1A and S6).
These results show that LAO1 is essential for the biosynthesis of IAA in Chlamydomonas.Although LAO1 mediates the deamination of L-Trp into IPyA, the first step in IAA biosynthesis, the subsequent step(s) may occur via a non-enzymatic decarboxylation of the keto acid by hydrogen peroxide 44 or may be catalyzed by an enzyme(s) (Figure 1G).We were unsuccessful in generating definitive support for either possibility (Table S2) so the precise mechanism for IPyA conversion to IAA remains to be elucidated.

IAA prevents Chlamydomonas from multiplying and attenuates chlorophyll degradation under N limitation
Since IAA controls cell growth in algae, 5,7,9,10,50 we asked whether this auxin has any effect on Chlamydomonas growth under the conditions for which we found IAA accumulation (i.e., under N limitation, in the absence of inorganic N).First, we tested the effect of L-Trp and LAO1 on Chlamydomonas growing on different N sources (Figure S1).Whereas growth on some amino acids like L-Ala and L-Trp is strictly dependent on LAO1, L-Arg can also be directly transported into the cell supporting growth independent of LAO1 41,51 (Figure S1A).Thus, to test the impact of LAO1, we compared the growth of WT and lao1 strains growing on media with L-Arg, supplemented with a range of L-Trp concentrations (Figure S1B).We observed that for WT cells, growth was improved at lower L-Trp concentrations up to 5 mM.However, this improvement was not observable at higher concentrations (10 mM) (Figure S1B).Growth of the lao1 mutant was not affected by L-Trp supplementation, however, suggesting that this concentration-dependent effect of L-Trp is due to LAO1 activity.We also tested the effect of L-Trp addition on cells growing on L-Ala as another potential LAO1-dependent N source; in this case, we only tested this using the WT strain because the lao1 mutant cannot grow on L-Ala (or L-Trp) (Figure S1A).We observed that higher concentrations of L-Trp inhibited growth, regardless of whether they were grown on L-Ala (Figure 2A) or not (Figure S1C), suggesting that the growth effects were L-Trp-specific.
Lastly, we examined the impact of L-Trp addition on WT and lao1 cells growing on ammonium, which can support growth independently of LAO1 and is also a strong repressor of LAO1 expression. 41In contrast to the phenotype observed in cells growing on L-Ala, no impact of L-Trp addition was observed for WT cells when grown on ammonium (Figure S1D).This result can be explained by the absence of LAO1 in ammonium and is consistent with the role of this enzyme in the concentration-dependent effect of L-Trp that we observed with L-Ala.To better understand the effect of LAO1-produced IAA on Chlamydomonas growth, cells were supplemented with different concentrations of L-Trp, IPyA, and IAA; this was conducted in the presence of L-Ala to ensure basal growth and that LAO1 is expressed (Figures 2A-2C).Note that given the presence of L-Ala, we did not expect the rate of IPyA/IAA accumulation in this experiment to be as high as the results shown in Figure 1 because LAO1 has a higher specific activity for L-Ala than for L-Trp. 40We initially tried a wider range of IAA concentrations (not shown) but chose to use a limited range of concentrations of IAA: from the minimum concentration that does not affect growth (12.5 mM IAA) to a maximum that reaches a level similar to that for 10 mM L-Trp (100 mM IAA).Similar concentrations of IPyA were used as with IAA to enable fair comparisons with IAA results.A slight increase in cell yield was observed at 1 mM L-Trp (Figure 2A).This could be explained by a slightly higher total N input and/or due to the production of IAA, which could have a stimulatory effect at low concentrations. 50Growth reduction was observed at higher concentrations of L-Trp, however, which could be attributed to the accumulation of IAA and other byproducts.Indeed, IAA concentrations higher than 10 mM had a clear inhibitory effect on culture cell density, with almost 70% inhibition at 100 mM IAA (Figure 2C).In contrast, similar levels of the intermediate IPyA did not affect growth (Figure 2B).This is consistent with the inability we observed of producing IAA directly from IPyA under these conditions (Table S2), but this may be due to the low stability of IPyA in solution 52,53 so these results must be interpreted cautiously.
The reduced cell density observed with increasing concentrations of extracellular IAA was correlated with an increase (up to four times) in chlorophyll content per cell (Figure 2C), which was not observed with IPyA (Figure 2B).This effect was also not observed with high levels of L-Trp (Figure 2A), possibly because additional intermediates produced from L-Trp deamination (e.g., hydrogen peroxide) may impair the Chlamydomonas wild-type (dark green circles) and lao1 null mutant (light green triangles) log-phase cells at 5310 6 cells/ml were incubated for 48 h in nitrogenfree medium supplemented with 5 mM L-Trp as a sole source of nitrogen.This is the minimum concentration at which we observed a significant growth reduction in the absence of any other N source available (Figure S1C).accumulation of chlorophyll.N limitation generally leads to chlorophyll degradation, 49 so the higher chlorophyll content per cell we observed in response to exogenous IAA could be explained by a reduction in chlorophyll degradation, or an increase in cell volume or the formation of palmelloids, [54][55][56] which must be IAA-specific and not merely LAO1-specific.To test this, we incubated concentrated, N-starved Chlamydomonas cultures with IAA and indeed found that the degradation of chlorophyll during N deprivation was reduced (Figures 2D-2F and S7) with no apparent effect on cell volume and/or palmelloid formation.In Chlamydomonas, chlorophyll degradation under N deprivation is linked to the mobilization of stored N to allow cells to duplicate one additional round before the complete cessation of growth. 49Here, we observed that despite being N-deprived, Chlamydomonas cultures significantly reduce cell multiplication rate in the presence of IAA, which may explain the increased chlorophyll content of these cells.

IAA production in Chlamydomonas facilitates a mutualistic interaction with Methylobacterium aquaticum
Because IAA mediates plant/algal-bacterial interactions, we asked whether IAA and LAO1 could play a role in the establishment of interactions of Chlamydomonas with the bacterial genus of Methylobacterium, given prior work demonstrating mutualistic tendencies of this taxa with Chlamydomonas under N-limiting conditions. 37Following-up on this work, we quantified the growth of Chlamydomonas in coculture with 10 different species of methylobacteria using L-Trp as the sole N source.We observed that coculturing with Methylobacterium spp., including M. aquaticum, improved algal growth (Figure S8).Naively, this growth promotion could simply be due to methylobacteria mineralizing L-Trp to provision Chlamydomonas with ammonium, which is more efficiently used by this alga than L-Trp as we similarly reported for L-proline. 37However, this seems unlikely because we observed no algal growth promotion of the lao1 knockout mutant by methylobacteria (Figure S8), suggesting that LAO1 is essential for this growth enhancement.Given the conversion of L-Trp to IAA and the inhibitory effects of IAA on Chlamydomonas, we hypothesized that methylobacteria may promote algal cell growth by reducing the levels of IAA accumulated in the media in coculture relative to Chlamydomonas in monoculture.Indeed, in coculture with M. aquaticum, there was no inhibition of growth by IAA and no effect on chlorophyll content (Figures 3B and 3C), and measured auxin levels were highly reduced (Figure 3D), strongly supporting a methylobacteria-mediated degradation of IAA.Because auxin concentrations were not reduced in bacterial monocultures nor in algal monocultures, both microbes are necessary for auxin to be eliminated from the media in a cooperative fashion.This effect was tested with nine other Methylobacterium spp.but was only observed with M. aquaticum, showing a species-specific interaction (cf. Figure S9).Importantly, coculturing with exogenously added IAA resulted in higher cell density for both microbes compared with the respective monocultures (Figures 3E-3G).Therefore, both microbes mutually benefited from the presence of each other.In this medium, acetate can serve as a source of carbon and energy for both the alga and the bacterium. 42,57However, no N source other than 500 mM IAA was added to the media to ensure N limitation, thus no large growth rates were expected.Because no detectable ammonium was produced by the bacterial monocultures on IAA (Table S3), Chlamydomonas most likely benefits from relief of IAA-induced inhibition through the bacterial degradation of this auxin.On the other hand, this observation strongly suggests that the bacterium, prompted by the presence of the alga, is feeding on IAA as an N source, allowing it to thrive in coculture.

DISCUSSION
The interactions between algae and heterotrophic bacteria play a fundamental ecological role controlling nutrient cycling and biomass production in their habitats. 58Studies have shown that bacteria can produce the phytohormone IAA to promote algal growth, 5 resembling the role of this molecule in plant-bacterial associations.However, auxin production by the algal partner has been largely overlooked or neglected, 5,12 despite the important ecological and economic impact this may cause in both aquatic and terrestrial ecosystems. 59,60Moreover, there has been no known genetic pathway to produce IAA in algae thus far.Here, we show a novel and unexpected mechanism of IAA production in the model alga Chlamydomonas under N-limiting conditions.Chlamydomonas can deaminate L-Trp extracellularly via the LAAO enzyme LAO1 to yield the keto acid IPyA, the major precursor for auxin biosynthesis in plants, bacteria, and fungi. 20A pathway for tryptophandependent IAA production involving an LAAO gene was recently discovered in the plant-benefiting bacterium Gluconacetobacter diazotrophicus encoded by a gene cluster containing L-amino acid oxidase, cytochrome c, and ridA genes. 47Chlamydomonas harbors a similar gene cluster that includes L-amino acid oxidase (LAO1) and a putative RidA gene (LAO2).We recently characterized a Chlamydomonas lao1 knockout mutant that lacks extracellular LAAO activity that we used here to evaluate the relevance of this enzyme for IAA production. 41e have shown that LAO1 is essential to produce IAA from L-Trp via an IPyA intermediate.Whereas this enzyme is most likely involved in the initial step of L-Trp deamination to yield IPyA, the conversion of IPyA into IAA may be achieved by a two-step pathway mediated by ipdC decarboxylase (via indole-3-acetaldehyde intermediate) in some bacteria or by the single-step YUCCA pathway (flavin monooxygenase-like enzyme) in plants. 20As in G. diazotrophicus, no homologs for any of these enzymes have been identified yet in Chlamydomonas, and thus, the nature of the conversion of IPyA into IAA in both organisms awaits experimental confirmation.This mechanism could involve a non-enzymatic decarboxylation of the keto acid by hydrogen peroxide, although our results do not support this.Alternatively, it may require an enzyme that relies on the presence of L-Trp and/or LAO1 to be expressed, as the addition of IPyA did not yield IAA in either of the two studies.However, this may be due to the low stability of IPyA in solution so these results must be interpreted cautiously.In addition to IPyA and IAA, kynurenic acid was found to accumulate in the algal media.This L-Trp metabolite can be produced via IPyA oxidation by free radicals. 61,624][65] Here, we demonstrated a LAO1-and L-Trpdependent mutualism between Chlamydomonas and M. aquaticum mediated by IAA, although other L-Trp metabolites like kynurenic acid may be responsible for mutualism with other Methylobacterium species and bacteria.Whether kynurenic acid production by Chlamydomonas is involved in mutualistic interactions with other microbes is unknown, but we believe this merits further investigation.
Previously limited to a single bacterial species, the role of LAAO enzymes in IAA production has been broadened here to include the microalga Chlamydomonas and may extend to other algae that harbor LAO1 homologs including members of Rhodophyta, Alveolata, Haptophyta, Heterokonta and Dinophyta. 41In support of this idea, the haptophyte alga Emiliania huxleyi harbors two putative LAO1 gene homologs 41 and shows a ratio of L-Trp conversion to IAA similar to what we found here in Chlamydomonas (16%-20% of L-Trp conversion). 17owever, based on existing genomic data, the LAO1 gene seems to be absent in most members of the green lineage (i.e., green algae and land plants) other than Chlamydomonas spp., so they must rely on alternative pathways for IAA biosynthesis. 41Higher levels for IAA production in Chlamydomonas have been reported relative to 23 other green algal species, 66 consistent with Chlamydomonas producing IAA using a different strategy compared with sister lineages.To date, transcriptomic and/or bioinformatic analyses appear to indicate that the most common putative pathway for IAA biosynthesis in microalgae, including Chlamydomonas, occurs via an indole-3-acetamide intermediate (reviewed by De Smet et al. 67 ).Although we have shown that LAO1 is crucial for IAA production under N limitation, alternative pathways that remain to be elucidated may be relevant under different conditions.
The LAO1 gene in Chlamydomonas was likely acquired through horizontal gene transfer, suggesting an adaptive advantage of having LAO1 in Chlamydomonas' native environment. 410][71] In this context, LAO1 could play a prominent role in scavenging N from a broad range of amino acids. 413][74] The accumulation of LAO1 keto acid byproducts as public goods in the phycosphere that are unused by the alga could have significant ecological roles that have been thus far neglected.Here, we have shown one example of how LAO1 can lead to extracellular IAA production with physiological and ecological consequences for the alga.
Although relatively low concentrations of IAA can improve algal growth, 50 we observed that the accumulation of high levels of IAA in the media arrests algal cell proliferation and chlorophyll degradation.6][77] An increase in photosynthetic performance by IAA has also been observed in the microalgae E. huxleyi and diatoms that harbor IAA-producing symbiotic bacteria. 5,17Similar effects have been observed in the chloroplasts of plants. 78This effect of IAA on the chlorophyll content is stronger in aged algal cultures and plant samples 17,78,79 and might be linked to a condition of stress. 80We considered the effect of IAA on chlorophyll levels in the context of N limitation as a stress condition that triggers LAO1 accumulation.Under mixotrophic conditions (i.e., in the presence of acetate), this alga prioritizes cellular respiration over photosynthesis. 49As a result, when N is limiting, pigments and other N-containing macromolecules involved in photosynthesis and chloroplast function are degraded first, presumably to enable Chlamydomonas cells to undergo an additional round of duplication. 49The accumulation of exogenous IAA by Chlamydomonas could function as a quorum-sensing-like signal molecule to decrease growth when the cell population is high and nutrient resources are limited. 12,18Preventing cell multiplication while avoiding the breakdown of the photosynthetic machinery during N limitation may be a hedge-betting strategy to capitalize on mutualistic cross-feeding interactions with other N-mineralizing microbes in the vicinity (e.g., Methylobacterium).These neighboring microbes may benefit from the provisioning of photosynthates like glycerol in exchange for more readily assimilable sources of N like ammonium that Chlamydomonas can use to resume growth. 37,81AA can be metabolized by rhizospheric bacteria, 82-85 some of which like Pseudomonas putida show chemotaxis toward the auxin. 85,86hus, IAA production by Chlamydomonas may attract and feed bacteria that could potentially be beneficially self-serving.8][89] Here, we demonstrate another mutualistic mode of interaction between Chlamydomonas and a Methylobacterium sp., mediated through IAA.M. aquaticum, induced or complemented by the presence of Chlamydomonas, can feed on IAA, which can ''release the brake'' on Chlamydomonas growth and facilitate the enhanced growth of both organisms.From the algal perspective, LAO1-mediated production of extracellular ammonium and keto acids may thus constitute a multifaceted strategy of (1) signaling, (2) waiting for partner feedback, (3) followed by mutualistic stimulation that may mirror a sort of call-and-response dynamic of plant-microbe interactions. 90o our knowledge, this is the first study to report an algal-dependent mechanism for IAA degradation in bacteria and IAA degradation by Methylobacterium spp. in general.Bacterial genes for IAA catabolism (iac) 91 appear to be absent from available Methylobacterium genomes but we believe that IAA degradation by M. aquaticum (involving a yet unidentified pathway) may cause a carbon/nitrogen metabolic imbalance that hampers its growth in the absence of the alga. 92During coculture, algal-derived photosynthates may restore carbon/nitrogen balance and enhance bacterial growth. 37We cannot rule out the possibility that other putative metabolites or proteins produced by the alga may also potentially facilitate bacterial degradation of IAA.Regardless, this observation supports that IAA-degrading bacteria can influence not only land plants but also algae. 91A previous study found that the haptophyte microalga E. huxleyi exhibits strain-specific differences in the production of IAA and susceptibility to infection by the pathogenic roseobacter Ruegeria sp.R11: while the non-IAA producer strain is resistant to roseobacter, the IAA-producing strain is susceptible to bacterial infection. 17This bacterial infection is accelerated by the addition of L-Trp to the coculture, which suggests that L-Trp-derived IAA production by the alga could enhance bacterial infection.However, because the levels of IAA in the algal cultures were shown to be reduced in coculture with roseobacter, the role of algal-derived IAA production was not pursued. 17Given the findings presented here showing that bacterial degradation of IAA can be triggered in the presence of algae, we speculate that in the presence of E. huxleyi, Ruegeria sp. may metabolize algal-derived IAA to achieve higher bacterial densities and potentially accelerate algal infection.Regardless, we believe that algal-dependent bacterial degradation of IAA may be relevant for many other bacterial species and their interactions with both algae and land plants.
Among the 10 Methylobacterium spp.tested in this work, M. aquaticum was significantly more efficient in degrading IAA in the presence of Chlamydomonas.This may underlie the basis for this particular bacterial species' broad ability to enhance the growth of early diverging plant lineages like green algae and moss. 37,935][96] Given niche overlap of algae and higher plants in soil and the ability of algae to ''dialogue'' with other microbes using the same auxin compounds that regulate land plants growth and development, we hypothesize that algae may play an underappreciated role in the recruitment of PGPB to the plant microbiome, let alone in modulating plant fitness through algal-plant interactions (Figure 4).Plant-plant interactions dramatically shape plant fitness, coexistence, life histories, and community assembly 36,97 ; we believe it is likely that the interactions of plants with earlier-diverging green lineages are also important and relevant to contemporary ecosystems. 98,99he role of microalgae as players in the plant microbiome has only recently started to be appreciated and their use to improve soil fertility, water preservation, and plant growth is now emerging as a promising approach for sustainable agriculture 48,59,[100][101][102][103][104] (see Alvarez et al. 105 for a recent review).For these new applications, understanding how and under what environmental conditions IAA is produced by rhizospheric and phyllospheric algae is of great interest.Bacterial degradation of IAA has been shown to be essential for rhizosphere colonization and plant growth promotion, 106 and similar to how LAAO-mediated auxin production is involved in plant growth promotion by G. diazotrophicus, 47 auxin production by other LAAO-containing microbes in the rhizosphere like Chlamydomonas could have an impact on plant growth and fitness.Our findings suggest that future studies should carefully consider the role that terrestrial algae play in plant microbiomes 48 and in the historical record of land plant evolution. 107

Article Conclusions
We provide genetic evidence that supports IAA production by the model alga Chlamydomonas via an unexpected LAAO-mediated pathway, which has thus far been restricted to a single bacterial species. 47This finding supports a new role for microbial LAAOs in auxin production that may be widespread in other algal lineages. 17,41We also show that IAA may act as an extracellular self-regulatory/quorum-sensing-like molecule to control cell multiplication and delay the breakdown of the photosynthetic machinery under inorganic N-limitation.This may be an ecological strategy to facilitate interactions with N-mineralizing bacteria.We demonstrate that the plant-and algal-benefiting bacteria Methylobacterium spp.can degrade extracellular levels of IAA generated by the alga in the presence of L-Trp and N limitation to alleviate inhibition of algal cell multiplication.Interestingly, these bacteria can feed on IAA only in the presence of the alga, revealing a new cooperative mode of auxin degradation that is induced and/or functionally complemented by the alga.This new mode suggests a role for algal-driven IAA production prompted by algal-bacterial interactions that may be relevant to plant-microbiome dynamics.Because Chlamydomonas and Methylobacterium are naturally found in the rhizosphere, their role in modulating IAA levels may impact plant fitness and could be exploited for crop improvement in sustainable agriculture.Overall, this work extends our understanding of auxin production and degradation by algae and In soil environments where tryptophan may be present due to plant exudation and microbial decay, bacterial indole-3-acetic acid (IAA) biosynthesis from this amino acid can promote plant growth.Chlamydomonas reinhardtii can also convert tryptophan into IAA using the extracellular enzyme LAO1.Accumulation of this auxin may result in algal growth arrest and in attracting beneficial PGPB bacteria.In the presence of the IAA-degrading bacterium Methylobacterium aquaticum, IAA is depleted, enhancing growth of both microorganisms.Metabolites exchanged between bacteria and algae could strengthen this mutualistic interaction. 37lgal-bacterial consortia under N-limitation, highlighting the potential for tri-partite interactions between rhizospheric algae, PGPB, and land plants.

Limitations of the study
The results of this study show that LAO1 is essential for the biosynthesis of IAA in Chlamydomonas.Although LAO1 is involved in the first step of IAA biosynthesis, namely L-Trp deamination, the subsequent step(s) for IPyA conversion to IAA remains unclear.Whereas algal growth on L-Trp was improved by four different Methylobacterium spp., IAA degradation could be attributed only to M. aquaticum in this work.Additional analyses of the metabolites from algal-bacterial cocultures using other Methylobacterium species would be beneficial in revealing the basis of other potential interactions.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:
To detect potential contamination, cell inocula were routinely checked by streaking on agar plates of TAP supplemented with yeast extract (2.5 g$l -1 ), incubated for 2 weeks at 23 C and examined under a light microscope.

METHOD DETAILS
Determination of L-tryptophan metabolization, and indole-3-pyruvic acid and indole-3-acetic acid biosynthesis by Chlamydomonas using HPLC Chlamydomonas wild-type and lao1 mutant were pre-cultured as described above to exponential phase.Cells were harvested by centrifugation for 2-3 min at 2,090 x g and washed three times with nitrogen-free TAP medium (T-N).Cells were subsequently incubated in T-N medium supplemented with 5 mM of L-Trp under continuous light and agitation (120 rpm).After 24 and 48 hours, cell-free supernatants were stored at -20 C until analyzed.L-Trp, indole-3-pyruvic acid and indole-3-acetic acid analysis was performed by the Chromatography Department staff at the Central Service for Research Support (SCAI) of the University of Co ´rdoba, using High-Performance Liquid Chromatography (HPLC) and UV-Vis detection.A mixture of L-Trp (Sigma-Aldrich, Spain), indole-3-pyruvic acid (Sigma, Spain) and indole-3-acetic acid (GoldBio, US) was included as standard.

LC-MS/MS analysis
Chlamydomonas wild-type cells were pre-cultured and incubated as described above in TAP-N medium supplemented with 5 mM of L-Trp under continuous light and agitation (120 rpm).After 24 and 48 and 96 hours, 300 ml of cell-free supernatants were stored at -20 C until analyzed.Freeze-dried extracts were resuspended in 2 ml of ultrapure water (Milli-Q A10 Advantage, MilliporeSigma, Burlington, MA) and applied to a Fisherbrand PrepSep C18 solid-phase extraction (SPE) column (FisherScientific, Pittsburgh, PA) that was pre-treated with 2 ml 100% HPLC grade methanol (A452-4, Fisher Scientific, Fair Lawn, NJ) followed by 2 ml ultrapure water and then 2 ml of 1 M HPLC grade acetic acid (AX0074-6, EMD Chemicals Inc., Gibbstown, NJ).The column was washed with 1 ml of 1 M acetic acid followed by 1 ml of 1% acetic acid.Analytes were then eluted using 2 ml of 100% methanol and evaporated until dried.UHPLC-MS/MS analysis of purified extracts was performed on an Orbitrap Exploris 240 instrument (Thermo Scientific, San Jose, CA) coupled to a Dionex Ultimate 3000 UHPLC system.Samples were loaded onto a PepMap 100 C18 column (0.3 mm 3 150 mm, 2 mm, Thermo Fisher Scientific).Separation of the samples was performed using mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) at a rate of 5 ml/min.The samples were eluted with a gradient consisting of 2% to 60% solvent B over 13 min, ramped to 95% B over 2 min, held for 8 min, and then returned to 2% B over 1 min and held for 8 min.All data were acquired in positive ion mode.Collision induced dissociation (CID) was used to fragment molecules, with an isolation width of 2 m/z units.The spray voltage was set to 3900 V, and the temperature of the heated capillary was set to 300 C. In CID mode, full MS scans were acquired from m/z 150 to 1800 followed by eight subsequent MS 2 scans on the top ten most abundant peaks.The orbitrap resolution for the MS1 scan was 60,000 and MS2 scans was 30,000.The expected mass accuracy based on external calibration was <3 ppm.Compound Discoverer 3.1 (Thermo Fisher Scientific, San Jose, USA) with the mzCloud database was used for metabolite identification, with mass tolerance set to 0.05 Da and retention time tolerance set to 0.2 min.A Fragment Ion Search (FISh) was subsequently performed on the compounds annotated by mzCloud.Compounds exhibiting a FISh score higher than 60 were considered as successfully identified compounds.Raw UHPLC-MS/MS files for these data have been deposited in the MassIVE database (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) with accession number MSV000092315.

Chlamydomonas cell growth tests
To test Chlamydomonas growth, cells were pre-cultured and washed as described above, placed in wells of a 48-well culture plate (BRANDplates, BrandTech Scientific, US) and incubated in fresh culture medium at an initial concentration of 0.2x10 6 cells/ml.In these growth assays L-alanine was supplemented in the medium as indicated in the figure captions to allow algal growth while ensuring N-limiting conditions and LAO1 activity.At the indicated times, cell concentrations were determined using 100-200 ml in a microcell counter (Sysmex F-500, Sysmed Inc, Europe).

Indole-3-acetic acid degradation tests
To determine microbial indole-3-acetic acid depletion from the medium, Chlamydomonas and Methylobacterium spp.mono-and co-cultures were initially pre-cultured independently as described above.After 2 days (exponential growth phase), cells were washed using T-N medium and incubated in fresh T-N medium supplemented with 500 mM of IAA under continuous light and agitation (120 rpm).Initial cell densities if algae and bacteria were set at 1.5x10 6 cells/ml and A 600 =0.01 (approximately 10 6 cells/ml), respectively.A negative control without inoculum was incubated to account for any potential abiotic degradation of IAA.After 1 week of incubation, the cell-free supernatants were collected and stored at -20 C subsequent IAA concentration measurements.For IAA analysis, 100 ml of freshly prepared Salkowski's reagent 113 (12 mg/ml FeCl 3 in 7.9 M H 2 SO 4 ) was added to the same volume of 1/5X diluted samples in flat-bottom 96-wells microplates and incubated for 30 min at room temperature (23-26 C) in the dark.A 540 absorbance was read using a microplate reader (iMarkTM, Bio-Rad).IAA was used as standard, although other indole acids including IPyA and indole-lactic acid could technically interfere.Calibration curves were included using 10-100 mM IAA in T-N medium.In these samples, ammonium content was measured using Nessler's reagent, 37 prepared mixing equal volumes of reagents A and B (MERCK 109011 and 109012, respectively).100-ml samples of cell-free supernatants were transferred to flat-bottom 96-wells microtiter plates.A volume of 100 ml of freshly prepared Nessler's reagent mixture was added, incubated for 2 min and A 410 was read using a microplate reader (iMark, Bio-Rad).Ammonium calibration curves containing at least ten points of known concentrations of NH 4 Cl, ranging from 50 to 1,000 mM, were included in every measurement and samples were diluted as needed.

Cell quantification by quantitative PCR
The simultaneous quantification of Chlamydomonas reinhardtii and Methylobacterium aquaticum cell number was inferred by the quantification of the algal-and bacterial-specific single-copy genes as described in Calatrava et al. 37 In brief, 213 bp of the Chlamydomonas centrin gene (Cre11.g468450.t1.2) were amplified using primers Cen1CreU and Cen1CreL and 239 bp of the Methylobacterium rpoB gene (Ma-q22A_c27070) were amplified using primers rpoBMaqU and rpoBMaqL.The sequences of these primers are indicated in the key resources table.For each qPCR run, 1 ml of each standard containing 1,010 copies/ml (centrin and rpoB) was serially 10-fold diluted, from 10 9 to 10 1 copies, and loaded in the same qPCR plate to quantify gene copies.qPCR was performed using SsoFast EvaGreen Supermix (Bio-Rad), and run and detected in MyiQTM2 (Bio-Rad) detection system.The conditions used for qPCR were: initial denaturation at 98 C for 2 min and 40 cycles of 5 s of denaturation at 98 C and 10 s of annealing and extension at 61 C.
The gDNA from the algal and bacterial samples was extracted as described in Calatrava et al. 37 One ml of culture was harvested by centrifugation for 5 min at 3,000 x g and the pelleted cells were resuspended in 800 ml of lysis buffer (50 mM Tris$HCl pH 8.0; 0.3 M NaCl; 5 mM EDTA, pH 8.0; 2% sodium dodecyl sulfate).Then, samples were frozen and stored at -80 C until processed for phenol-chloroform extraction.Samples were thawed at 4 C and extracted using an equal volume of a phenol solution containing phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 50 mM Tris$HCl, and vortexing vigorously for 1 min.Then, the samples were centrifuged for 5-10 min at 15,000 x g to separate both phases.The aqueous phase was transferred to a new tube and the extraction step with phenol solution was repeated for 2-3 times until no interphase was observed.Then, a last extraction step with chloroform was performed.DNA precipitation was achieved with 0.9 volumes of isopropanol and incubating for 1 hour at room temperature and then centrifuged for 30 min at 15,000 x g.The obtained gDNA was treated with RNase H (Promega).Nucleic acids concentration was quantified spectrophotometrically using NanoDropä (Thermo Scientificä).

QUANTIFICATION AND STATISTICAL ANALYSIS
Data represent averages G Standard Deviation.T-tests were performed using GraphPad Prism 6 with a<0.05 (*), a<0.005 (**); a<0.001 (***), and three biological replicates (n=3) unless otherwise indicated in the figure caption.Data show a representative experiment and biological replicates were inoculated from independent pre-cultures and run in parallel.

Figure 1 .
Figure 1.L-amino oxidase (LAO1) plays a critical role in indole-3-acetic acid (IAA) production from L-tryptophan (L-Trp) in ChlamydomonasChlamydomonas wild-type (dark green circles) and lao1 null mutant (light green triangles) log-phase cells at 5310 6 cells/ml were incubated for 48 h in nitrogenfree medium supplemented with 5 mM L-Trp as a sole source of nitrogen.This is the minimum concentration at which we observed a significant growth reduction in the absence of any other N source available (FigureS1C).(A and D) L-Trp, (B and E) indole-3-pyruvic acid (IPyA), and (C and F) IAA were quantified in the cell-free supernatants by HPLC; the identity of these compounds was confirmed with LC-MS/MS.The MS2 match was performed using the Fragment Ion Search function of Compound Discoveror 3.1 (Thermo Fisher Scientific, San Jose, USA).Green dots represent MS2 matchings for L-Trp (B), IPyA (D), and IAA (F).(G) In the periplasm, LAO1 and LAO2/RIDA (Reactive Intermediate/Imine Deaminase A) deaminate extracellular L-Trp to produce the a-keto acid IPyA, which is decarboxylated to IAA by means of a yet unidentified mechanism (dashed arrows).
Figure 1.L-amino oxidase (LAO1) plays a critical role in indole-3-acetic acid (IAA) production from L-tryptophan (L-Trp) in ChlamydomonasChlamydomonas wild-type (dark green circles) and lao1 null mutant (light green triangles) log-phase cells at 5310 6 cells/ml were incubated for 48 h in nitrogenfree medium supplemented with 5 mM L-Trp as a sole source of nitrogen.This is the minimum concentration at which we observed a significant growth reduction in the absence of any other N source available (FigureS1C).(A and D) L-Trp, (B and E) indole-3-pyruvic acid (IPyA), and (C and F) IAA were quantified in the cell-free supernatants by HPLC; the identity of these compounds was confirmed with LC-MS/MS.The MS2 match was performed using the Fragment Ion Search function of Compound Discoveror 3.1 (Thermo Fisher Scientific, San Jose, USA).Green dots represent MS2 matchings for L-Trp (B), IPyA (D), and IAA (F).(G) In the periplasm, LAO1 and LAO2/RIDA (Reactive Intermediate/Imine Deaminase A) deaminate extracellular L-Trp to produce the a-keto acid IPyA, which is decarboxylated to IAA by means of a yet unidentified mechanism (dashed arrows).

Figure 2 .
Figure 2. IAA arrests cell multiplication and attenuates chlorophyll degradation in nitrogen-limited Chlamydomonas (A-C) Impact of exogenously added tryptophan (L-Trp), indole-3-pyruvic acid (IPyA), and indole-3-acetic acid (IAA) on Chlamydomonas growth in the presence of L-alanine (L-Ala).Wild-type cells at an initial concentration of 0.2310 6 cells/mL were grown for three days on L-Ala (4 mM) as an N source (to enable algal growth under N-limiting conditions and ensuring LAO1 expression; see STAR Methods) in the presence of the indicated concentrations of (A) L-Trp, (B) IPyA, or (C) IAA.Cell culture densities are indicated as solid lines and chlorophyll content per cell as dotted lines.(D-F) Impact of IAA in Chlamydomonas during N deprivation.(D) Cell density and (E) chlorophyll content during N deprivation (in the absence of any assimilable N source).Wild-type cells at 10 6 cells/ml were incubated in N-free media (-N) or supplemented with 500 mM of IAA (IAA).Data are averages of 3 biological replicates with error bars depicting standard deviations.Asterisks indicate statistically significant differences compared with the control without IAA (t test: n = 3; *a < 0.05; **a < 0.005; ***a < 0.001).(F) A representative culture flask of each condition in panels (D) and (E) was imaged at the start, after one day, and after five days; cell pellets shown were harvested by centrifuging 0.5 mL of the cultures after five days of incubation.

Figure 3 .
Figure 3. Methylobacterium aquaticum reduces IAA levels in Chlamydomonas cultures to relieving algal inhibition of cell multiplication and chlorophyll degradation (A) Algal cell density and (B) chlorophyll content per cell were determined initially (0 days) and after five days of incubation for Chlamydomonas monocultures (Cre) and Chlamydomonas-M.aquaticum co-cultures (Cre-Maqu) in N-free medium (-N) supplemented with 500 mM IAA (IAA).Initial cell concentrations were 10 6 cells/ml for Chlamydomonas and A 600 of 0.01 for M. aquaticum (approximately 10 6 cells/mL).Chlamydomonas monocultures correspond to the same dataset as in Figures 2D and 2E and are represented here as a reference for algal monocultures.(C) Chlamydomonas (Cre) monocultures, M. aquaticum (Maqu) monocultures, and co-cultures (Cre-Maqu) were incubated on N-free media supplemented with 500 mM of IAA for five days.Indoles concentration in the cell-free media were determined using the Salkowski reagent (see STAR Methods).(D) Algal and bacterial cell densities after eight days of growth on 500 mM of IAA in coculture or monoculture were quantified using qPCR of single-copy genes specific for Chlamydomonas (centrin) or M. aquaticum (rpoB) (see STAR Methods).Data shown are averages of 3 biological replicates with error bars depicting standard deviations.Asterisks indicate statistically significant differences compared with the control comparison (t test: n = 3; *a < 0.05; **a < 0.005; ***a < 0.001).(E) Chlamydomonas-M.aquaticum cocultures were imaged using a light microscope under conditions without a nitrogen source (-N) and (F) supplemented with 500 mM IAA (IAA) after five days.

Figure 4 .
Figure 4. Proposed role of auxin-mediated mutualistic interactions between Chlamydomonas, Methylobacterium, and land plants For a Figure360 author presentation of this Figure 4, see https://doi.org/10.1016/j.isci.2023.108762.In soil environments where tryptophan may be present due to plant exudation and microbial decay, bacterial indole-3-acetic acid (IAA) biosynthesis from this amino acid can promote plant growth.Chlamydomonas reinhardtii can also convert tryptophan into IAA using the extracellular enzyme LAO1.Accumulation of this auxin may result in algal growth arrest and in attracting beneficial PGPB bacteria.In the presence of the IAA-degrading bacterium Methylobacterium aquaticum, IAA is depleted, enhancing growth of both microorganisms.Metabolites exchanged between bacteria and algae could strengthen this mutualistic interaction.37Moreover, we imagine three-way algal-plant-bacterial associations whereby algal-derived IAA not only benefits the alga but promotes plant-bacterial symbioses and modulates plant physiology directly.Figure created using BioRender.comand ChemDraw 20.1.[*] indicates interactions or processes shown in this work.Numbers in square brackets correspond to references: [1] Vallon et al. 40 ; [2] Cox et al. 1,4,8 ; [3] Rico-Jime ´nez et al. 85 ; [4] Kravchenko et al. 70 ; [5] Moe LA 108 ; [6] Backer et al.109 Figure 4. Proposed role of auxin-mediated mutualistic interactions between Chlamydomonas, Methylobacterium, and land plants For a Figure360 author presentation of this Figure 4, see https://doi.org/10.1016/j.isci.2023.108762.In soil environments where tryptophan may be present due to plant exudation and microbial decay, bacterial indole-3-acetic acid (IAA) biosynthesis from this amino acid can promote plant growth.Chlamydomonas reinhardtii can also convert tryptophan into IAA using the extracellular enzyme LAO1.Accumulation of this auxin may result in algal growth arrest and in attracting beneficial PGPB bacteria.In the presence of the IAA-degrading bacterium Methylobacterium aquaticum, IAA is depleted, enhancing growth of both microorganisms.Metabolites exchanged between bacteria and algae could strengthen this mutualistic interaction.37Moreover, we imagine three-way algal-plant-bacterial associations whereby algal-derived IAA not only benefits the alga but promotes plant-bacterial symbioses and modulates plant physiology directly.Figure created using BioRender.comand ChemDraw 20.1.[*] indicates interactions or processes shown in this work.Numbers in square brackets correspond to references: [1] Vallon et al. 40 ; [2] Cox et al. 1,4,8 ; [3] Rico-Jime ´nez et al. 85 ; [4] Kravchenko et al. 70 ; [5] Moe LA 108 ; [6] Backer et al.109

TABLE
EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS d METHOD DETAILS B Determination of L-tryptophan metabolization, and indole-3-pyruvic acid and indole-3-acetic acid biosynthesis by Chlamydomonas using HPLC B LC-MS/MS analysis B Chlamydomonas cell growth tests B Chlorophyll determination B Indole-3-acetic acid degradation tests B Cell quantification by quantitative PCR d QUANTIFICATION AND STATISTICAL ANALYSIS d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d containing 8 mM of ammonium chloride at 23 C under continuous light and agitation (120 rpm).Methylobacterium strains used in this work are summarized in the key resources table.Bacterial cells were pre-cultured in Methylobacterium Medium (MeM)